Conjugates of antibodies with drugs, radioisotopes, and proteins have been widely investigated, and a range of chemical approaches are available and widely used. Conjugation is normally carried out to amino acid side chains, for example to lysine residues, in a random fashion such that a distribution of chemically modified species is found in each preparation of conjugate, where different positions in the antibody may be modified each time. Although successfully used, this approach has several disadvantages. These include the fact that amino acids important for function of the antibody, for example in antigen binding or Fc receptor binding, may be modified and consequently functionality of the antibody may be modified or lost. In addition, the heterogeneity of the antibody conjugate in which a population of molecules exists with different side chains modified complicates analysis and makes it difficult to ensure that each preparation contains the same distribution of modified species.
A potential improvement to conjugation is to attach the drug or other molecule at a specific site, which is identical each time. This can be designed such that attachment to the site does not interfere with antibody functional properties, and allows simplified analysis and quality control of conjugate preparations. A number of approaches have been used to accomplish this either using naturally occurring sites in the antibody molecule or by specifically introducing additional sites through antibody engineering.
The generation of free cysteines by selective reduction of the hinge region has been used for the attachment of thiol reactive compounds to both IgG and antibody fragments, for example, to attach fluorescent compounds (Packard et al., Biochem. 25, 3548-3552 (1986)), for attachment of chelators that can be used for site-specific radiolabelling (King et al., Cancer Res., 54, 6176-6185 (1994)), and for drug attachment (Doronina et al., Nature Biotechnol. 21, 778-784 (2003)). Disadvantages of this approach include the reduction of disulfide bonds which are important for maintenance of the native antibody structure. This may have detrimental effects on the functionality or stability of the resulting conjugate. Also, as several disulfide bonds are present in the antibody molecule, including two in the hinge region for human IgG1, one attaching each light chain to heavy chain, and one internal disulfide in each folded immunoglobulin domain, there remains potential heterogeneity in the conjugate produced.
The Fc region carbohydrate also provides a natural specific attachment site for IgG molecules. The carbohydrate is usually modified by periodate oxidation to generate reactive aldehydes which can then be used to attach reactive amine containing compounds by Schiff base formation. As the aldehydes can react with amine groups, reactions are carried out at low pH so that the lysine residues are protonated and unreactive. Hydrazide groups are most suitable for attachment to the aldehydes generated since they are reactive at low pH to form a hydrazone linkage. The linkage can then be further stabilized by reduction with sodium cyanoborohydride to form a hydrazine linkage (Rodwell et al., Proc. Natl. Acad. Sci. (USA) 83, 2632-2636 (1986)). The disadvantages of this approach are the relatively harsh conditions required which can damage and aggregate some antibody molecules. Methionine residues present in some antibody variable regions may be particularly susceptible to oxidation by periodate which can lead to loss of antigen binding avidity. In some cases histidine or tryptophan residues might also be affected.
Antibody engineering can be used to introduce specific attachment sites into antibody molecules, and this can be incorporated as part of the design of an engineered molecule. Extra cysteine residues can be introduced onto the surface of antibody constant domains to provide a specific attachment site without the need to disrupt native disulphide bonds. Introduction of specific cysteine residues in the CH1 domain of the IgG heavy chain has been shown to result in sites to which ligands can be attached without any loss of antigen binding (Lyons et al., Protein Engin. 3, 703-708 (1990)). These mutations can be used to produce site-specifically labeled IgG or Fab antibody fragments (Bodmer et al., U.S. Pat. No. 5,219,996). Similar work to produce site-specific drug conjugates has recently been reported by Genentech (Eigenbrot et al., U.S. Patent Publication No. 2007/0092940).
Mutations in the Fc region of the antibody have also been explored. Substitution of a serine residue near the C-terminus of the CH3 domain (Ser444) to cysteine resulted in the production of IgG dimer of a chimeric human IgG1. This mutation resulted in 50% of the molecules forming dimeric IgG (Shopes, 1992). The same mutation introduced into a humanized IgG1 also resulted in the formation of IgG dimers (Caron et al., J. Exp. Med. 176, 1191-1195 (1992)). An alternative mutation in the Fc region at position 442 has also been generated and used for site-specific attachment (Stimmel et al., J. Biol. Chem. 275, 30445-30450 (2000)).
Antibody fragments such as single-chain Fv and diabodies have been engineered in which an extra cysteine residue is added at the C-terminus of the molecule (e.g. Cumber et al., J. Immunol. 149, 120-126 (1992); King et al, Cancer Res., 54, 6176-6185 (1994); L1 et al., Bioconjugate Chem. 13, 985-995 (2002); Yang et al., Protein Engineering 16, 761-770 (2003); Olafson et al., Protein Engineering Design & Selection, 17, 21-27 (2004)).
Alternative methods for site-specific attachment include the introduction of extra glycosylation sites to allow attachment via periodate oxidation. Some antibody light chains have an unusual natural glycosylation site, and thus the light chain has been used as a site to introduce a glycosylation site into antibodies which do not normally have a carbohydrate attached to the light chain (Leung et al., J. Immunol. 154, 5919-5926 (1995)). A third engineering strategy is to introduce extra lysine residues into the surface of the constant region domains (Hemminki et al., Protein Engin. 8, 185-191 (1995)). Although this approach does not introduce a unique labeling site, lysine reactive reagents are more likely to modify the antibody at the increased concentration of lysine residues in the constant region resulting in the retention of more antigen binding reactivity.
A more specialized approach is the use of reverse proteolysis to attach reagents specifically at the C-terminus of Fab′ heavy chains (Fisch et al., Bioconj. Chem. 3, 147-153 (1992)). After production of a F(ab′)2 fragment by the protease lysyl endopeptidase, experimental conditions can be altered such that the same protease working in reverse is capable of the specific attachment of carbohydrazide groups to the C-terminus of the F(ab′)2 heavy chains. These carbohydrazide groups could then be used as an attachment point for a radiolabelled chelator reacting via an aldehyde group to form a hydrazone linkage.
Despite the background art described above, there remains a need to conjugate partner molecules (e.g. a drug or toxin) to intact IgG molecules, which are better characterized and more stable than antibody fragments. The potential benefits of attachment of partner molecules to the IgG instead of antibody fragments include retention of Fc region dependent effector functions, such as Fc receptor-dependent ADCC and phagocytosis, and also retention of the FcRn binding site which allows a long serum half-life to be maintained. As described in detail below, the instant invention satisfies this need.